J Arthropod-Borne Dis, September 2020, 14(3): 270–276 L Ganushkina et al.: Detection of the … Original Article Detection of the Invasive Mosquito Species Aedes (Stegomyia) aegypti and Aedes (Hulecoeteomyia) koreicus on the Southern Coast of the Crimean Peninsula Lyudmila Ganushkina1; Alexander Lukashev1; *Ivan Patraman1; Vladimir Razumeyko2; Еlena Shaikevich1,3 1Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, Sechenov First Moscow State Меdical University, Moscow, Russia 2Department of Ecology and Zoology Taurida Academia, Vernadsky Cremian Federal University, Simferopol, Republic of Crimea 3Vavilov Institute of General Genetics Russian Academy of Sciences, Moscow, Russia (Received 24 Sep 2018; accepted 05 Sep 2020) Abstract Background: The incidence and area of arbovirus infections is increasing around the world. It is largely linked to the spread of the main arbovirus vectors, invasive mosquito of the genus Aedes. Previously, it has been reported that Aedes aegypti reemerged in Russia after a 50-year absence. Moreover, in 2011, Ae. albopictus was registered in the city of Sochi (South Russia, Black Sea coast) for the first time. In 2013, Asian Ae. koreicus was found in Sochi for the first time. Methods: Mosquitoes were collected using the following methods: larvae with a dip net, imago on volunteers and using bait traps. The mosquitoes were identified using both morphology and sequencing of the second internal transcribed spacer of the nuclear ribosomal RNA gene cluster. Results: In August 2016, Ae. koreicus larvae and imago and a single male of Ae. aegypti were found on the southern coast of the Crimean Peninsula, where they were not registered before. Newly obtained DNA sequences were registered in GenBank with the accession numbers MF072936 and MF072937. Conclusion: Detection of invasive mosquito species (Ae. aegypti and Ae. koreicus) implies the possibility of their area expansion. Intensive surveillance is required at the Crimean Peninsula to evaluate the potential for the introduction of vector-borne diseases. Keywords: Mosquito monitoring; Arbovirus vectors; Aedes aegypti; Aedes koreicus; DNA Introduction At present, the invasive species Aedes (Steg- of Crimea, which has a climate very similar to omyia) aegypti, Aedes (Stegomyia) albopictus Caucasus. In 2010, an entomological survey in and Aedes (Hulecoeteomyia) koreicus are spread- the southern coast of Crimea found 15 blood- ing across the world and have been registered sucking mosquito species, namely three spe- in Caucasian coast of the Black Sea in south- cies of the genus Anopheles (An. claviger, An. ern Russia since 2011 (1-4). All of them are maculipennis, An. plumbeus), 3 species of the known as vectors of human pathogens (4). In- genus Culex (Cx. hortensis, Cх. pipiens, Cх. vasive Aedes species have adapted its habitat territans), five species of the genus Aedes (Ae. preferences to human made containers, such refiki, Ae. cantans, Ae. dorsalis, Ae. cataphylla, as water storage tanks, discarded tires and jars, Ae. geniculatus), three species of the genus and water-filled pots (4). Due to the changing Culiseta (Cs. annulata, Cs. longiareolata, Cs. climate and the introduction of artificial hatch- morsitans) and Uranotaenia unguiculata (5). ing habitats, it was expected that these mosquito The coastal area of Crimea, especially the south- species would also spread to the southern coast ern coast, is densely populated and attracts ap- 270 *Corresponding author: Dr Ivan Patraman, Email: http://jad.tums.ac.ir [email protected] Published Online: September 30, 2020 J Arthropod-Borne Dis, September 2020, 14(3): 270–276 L Ganushkina et al.: Detection of the … proximately five million tourists each year. genes (8) were used for amplification of the se- The goal of the present study was to survey cond internal transcribed spacer (ITS2) region the southern coast of Crimea on the presence of rRNA genes. Amplification products were of invasive mosquito species. visualized in 1% agarose gel and purified from the gel using a clean-up kit (Evrogen, Mos- Materials and Methods cow). Sequencing was performed using ABI PRISM® 310 Genetic Analyzer with BigDye® Terminator kit (Applied Biosystems, USA). Mosquito collection was carried out in Au- The obtained DNA sequences were registered gust, 2016. Mosquito sampling sites are shown in GenBank (accession numbers MF072936 in figure 1. The larvae were collected with a and MF072937). The internet resource dip net, while the imago were collected either https://www.gismeteo.ru/diary/ was used for on volunteers using sucking tubes or “Electro- climate analysis. frog” bait traps (LMD-Komplekt Plus, Russia). Mosquito sampling sites had different con- ditions. Priboy vacation resort (Number 1 in Results Table 1, Fig. 1) is located 300–500 meters to the northwest of Sakskoe Lake, one kilometer Imago and larvae of eight mosquito spe- to the east is the resort city Saki, a town with cies, 661 individuals in total, were collected well-developed residential areas. Frunzenets on the Crimean Black Sea coast and in Sim- vacation resort (Number 2 in Table 1, Fig. 1) feropol (Fig. 1, Table 1). is located one kilometer away from a village In Simferopol, Cx. pipiens was collected in on the shores of Lake Bagaily (Number 7 in water storage tanks. In Lesnoy Village (№6 in Table 1, Fig. 1), which were overgrown with Table 1, Fig. 1), located close to the town of reeds and the inshore depth reached 1 meter. Sevastopol, mosquitoes were collected in an Mosquito larvae were collected in the thickets artificial concrete basin with an area of 23m2 of the reeds. Departure of imago from the lake and a depth of 0.5m. In this artificial basin, was registered. Imagoes were collected in the the larval density per 1m2 was the highest: Ae. peak mosquito activity period from 8:30PM koreicus – 238 individuals; Cs. longiareolata to 9:30PM. – 256; and Cx. pipiens – 17. The mosquitoes were identified by morpho- Among them, one male Ae. aegypti was dis- logical key (7) and DNA sequencing. The most covered at Priboy vacation resort. Morpholog- reliable characteristics for Ae. koreicus larvae ical identification was confirmed by DNA se- were the form and number of сomb scales on quencing. This Ae. aegypti ITS2 DNA sequence the lateral view of larval abdominal segment was deposited to GenBank (accession number (Fig. 2A), position of the siphonal tuft and the MF072936). The ITS2 nucleotide sequence of number of the pectin teech (Fig. 2B). Imago the Crimean Ae. aegypti was identical to the Ae. koreicus show a black and white pattern one from Abkhazia, 2012 (MH142316) and due to the presence of white scale patches on Adler (Russia), 2013 (MH142319). There was a black background and have clear longitudi- one nucleotide substitution in ITS2 (99% nal lines on the scutum (Fig. 2C). Two narrow identity) between Ae. aegypti found in Crimea white dorsocentral stripes are characteristic for (MF072936) and Ae. aegypti sequences regis- Ae. aegypti (Fig. 2D). tered in GenBank: Rockefeller strain KF471581- DNA extraction was performed using Dia- KF471583; JX423805 and JX423807 from toms DNA prep kit (Isogen, Moscow). The Saudi Arabia; KU497616 from China; and Encyclo PCR kit (Evrogen, Moscow) and pri- KF471579 from New Caledonia. mers complementary to 5.8S and 28S rRNA Larvae of the species Ae. koreicus were found 271 http://jad.tums.ac.ir Published Online: September 30, 2020 J Arthropod-Borne Dis, September 2020, 14(3): 270–276 L Ganushkina et al.: Detection of the … close to Sevastopol in Lesnoy Village (Table In addition to these two invasive species, 1), in an artificial concrete basin. Aedes kore- representatives of six autochthonous mosquito icus imago were found next to this pond. The species were registered in the studied area. Ima- sequence of the ITS2 genome region of Ae. goes sampled at all locations were mainly rep- koreicus larvae found in Crimea was identical resented by the species Cx. pipiens, Cx. mod- to the DNA sequence of Ae. koreicus from estus, and a few individual Coquillettidia richi- Belgium (KF471636 and JF430391) and Sochi ardii and Ae. caspius (Table 1). Culiseta longi- (HG763830). The Crimean Ae. koreicus DNA areolata and Cx. pipiens larvae were found in sequence was deposited in GenBank with the Lesnoy Village (Table 1). accession number MF072937. Fig. 1. Map of Crimean Peninsula with the mosquito sampling locations indicated as in Table 1. The map of Europe with the current known established populations of Aedes koreicus and Ae. aegypti in Europe (ECDC; 2020. Available from: https://ecdc.europa.eu/en/disease-vectors/surveillance-and-disease-data/mosquito-maps), Crimean Peninsula indicated with a circle. Locations of Ae. aegypti collection are shown with a square, Ae. koreicus - with a star. Fig. 2. Diagnostic characters of Aedes koreicus larva (A, B) and female (C), Ae. aegypti male (D) 272 http://jad.tums.ac.ir Published Online: September 30, 2020 J Arthropod-Borne Dis, September 2020, 14(3): 270–276 L Ganushkina et al.: Detection of the … Table 1. Composition of adults and larvae of 661 collected mosquitoes Number Sample site Coordinates Mosquito species on the Ae. Ae. Ae. Cx. Cx. Cx. Cq. Cs. map aegypti koreicus caspius pipiens modestus hortensis richiardii longiareolata 1 Priboy 45°13'05″N 33°52'22″ E 1♂ 3♀ 154♀ 5♂ 157♀4♂ 11♀ 2 Frunzenets 45°02′37″N 33°63′28″Е 1♀ 52♀3♂ 90♀3♂ 2♀ 3 Evpatoria 44°12'00″N 33°21'30″E 26♀ 21♀ 4 Koktebel 44°57'35″N 35°14'52″E 3♀ 3♀ 5 Simferopol 44°56'53″N 34°06'15″E 12♀10L 6 Sevastopol 44°43′55″N (Lesnoy 33°34′55″E 3♀29L 2L 33L Village) 7 Lake 45°02′37″N Bagaily 33°63′28″Е 8L 16L 9L Total number of individuals 1 32 4 275 294 9 13 33 Discussion Screening of mosquitos on the south coast to populated areas located near woods.